JP6021647B2 - Method for production and purification of recombinant lysosomal alpha-mannosidase - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to a method for purifying recombinant α-mannosidase, a method for producing α-mannosidase, a composition containing α-mannosidase, the use of the composition as a medicament, and a medicament for treating α-mannosidosis. And the method of treating α-mannosidosis and / or reducing the symptoms of α-mannosidosis.

Background of the Invention
α-Mannosidosis α-Mannosidosis is a recessive autosomal disorder that occurs at a frequency of 1 / 1,000,000 to 1 / 500,000 worldwide. Mannosidosis is found in all ethnic groups in Europe, America, Africa, and Asia. This disease has been detected to a similar extent in all countries that provide high-grade diagnostic services for lysosomal storage disorders. Although they appear born as healthy people, the symptoms of the disease progress. α-Mannosidosis exhibits clinical diversity in the range of very severe to very mild forms. Typical clinical symptoms are mental retardation, skeletal changes, immune system disorders that cause recurrent infections, hearing impairments, which are often typical facial features such as coarse faces. Accompanied by protruding forehead, flat nose bridge, small nose, wide mouth. In its most severe case (mannosidosis type 1), children suffer from hepatosplenomegaly and die within the first year of life. This premature death is believed to be caused by a severe infection resulting from the immunodeficiency caused by the disease. In milder cases (mannosidosis type 2), patients usually reach adult age. Because patients have a weak skeleton, they need a wheelchair between the ages of 20 and 40. This disease causes diffuse brain dysfunction, and in many cases the low mental ability that results in it excludes everything but the most basic skills such as simple reading and writing. These problems associated with hearing loss and other clinical signs interfere with the patient's independence and, as a result, require lifelong care.

Lysosomal α-mannosidase α -mannosidosis results from a deficient activity of lysosomal α-mannosidase (LAMAN, EC 3.2.1.24). This disease is characterized by excessive intracellular accumulation of mannose-rich oligosaccharides, ie oligosaccharides having α1,2-, α1,3- and α1,6-mannosyl residues at the non-reducing end. These oligosaccharides arise primarily from lysosomal degradation of glycoproteins containing N-linked oligosaccharides. However, some arise from glycoproteins with misfolding that have been redirected to the cytosol due to catabolism and degradation by the proteasome of dolichol-linked oligosaccharides (Hirsch et al. EMBO J. 22, 1036-1046, 2003 (non- Patent Document 1) and Saint-Pol et al. J. Biol. Chem. 274, 13547-13555, 1999 (Non-Patent Document 2)). Its lysosomal accumulation is observed in a wide range of cell types and tissues, including neurons in the whole brain region. LAMAN is an exoglycosidase that hydrolyzes these terminal non-reducing α-D-mannose residues in α-D-mannosides from the non-reducing end in the stepwise degradation of N-linked glycoproteins (Aronson and Kuranda FASEB J 3: 2615-2622. 1989 (non-patent document 3)). The human precursor enzyme is synthesized as a 1011 amino acid single chain polypeptide containing a 49 residue signal peptide. This precursor is proteolytically processed into lysosomes into three major glycopeptides of 15, 42 and 70 kD to become mature enzymes. The 70 kD glycopeptide is further processed into three subunits linked by disulfide bridges (Berg et al. Mol. Gen. and Metabolism 73, 18-29, 2001, Nilssen et al.). al. Hum. Mol. Genet. 6, 717-726. 1997 (non-patent document 5)).

Lysosomal α-mannosidase gene
The gene encoding LAMAN (MANB) is located on chromosome 19 (19cen-q12) (Kaneda et al. Chromosoma 95: 8-12. 1987 (Non-patent Document 6)). MANB consists of 24 exons spanning 21.5 kb (GenBank action number U60885-U60899; Riise et al. Genomics 42: 200-207, 1997 (non-patent document 7)). The LAMAN transcript is >> 3,500 nucleotides (nts) and contains an open reading frame encoding 1,011 amino acids (GenBank U60266.1).

  Cloning and sequencing of the human cDNA encoding LAMAN has been published in three publications (Nilssen et al. Hum. Mol. Genet. 6, 717-726. 1997); Liao et al. J. Biol. Chem. 271, 28348-28358. 1996 (Non-patent document 8); Nebes et al. Biochem. Biophys. Res. Commun. 200, 239-245. 1994 (Non-patent document 9)). Oddly enough, the three sequences are not identical. Compared to the sequence of Nilssen et al (accession number U60266.1), Liao et al. And Nebes et al. Found changes from TA to AT at positions 1670 and 1671 resulting in substitution of valine to aspartic acid. It was done. A change from C to A at position 1152 was also found that did not cause any change in the amino acid sequence.

Diagnosis of α-mannosidosis is currently available for clinical evaluation, detection of urinary mannose-rich oligosaccharides and direct measurement of α-mannosidase activity in various cell types such as leukocytes, fibroblasts and amniotic cells. (Chester et al., In: Durand P, O'Brian J (eds) Genetic errors of glycoprotein metabolism. Edi-Ermes, Milan, pp 89-120. 1982; Thomas and Beaudet. In: Scriver CR, Beaudet AL, Sly WA, Valle D (eds) The metabolic and molecular bases of inherited disease. Vol 5. McGraw-Hill, New York, pp 2529-2562. 1995 (Non-patent Document 11)).

  In many cases, the diagnosis is often made late in the course of the disease, since the symptoms are initially mild and difficult to biochemically diagnose. It is clear that patients and their families will benefit greatly from early diagnosis.

Animal model α-mannosidosis has been reported in cattle (Hocking et al. Biochem J 128: 69-78. 1972 (Non-patent Document 12)) and cats (Walkley et al. Proc. Nat. Acad. Sci. 91: 2970- 2974, 1994 (Non-Patent Document 13)) and guinea pigs (Crawley et al. Pediatr Res 46: 501-509, 1999 (Non-Patent Document 14)). Recently, a mouse model was created by specific disruption of the α-mannosidase gene (Stinchi et al. Hum Mol Genet 8: 1366-72, 1999 (Non-patent Document 15)).

  As in humans, α-mannosidase appears to be caused by specific mutations in the gene encoding lysosomal α-mannosidase. Berg et al. (Biochem J. 328: 863-870. 1997) (Non-Patent Document 16) reports the purification of feline liver lysosomal α-mannosidase and determination of its cDNA sequence. The active enzyme consists of three polypeptides and their molecular weights have been reported to be 72, 41 and 12 kD. Like the human enzyme, the feline enzyme has been demonstrated to be synthesized as a single-chain precursor of a 957 amino acid polypeptide chain that is cleaved into three polypeptides of the mature enzyme followed by a putative signal peptide of 50 amino acids. . Its deduced amino acid sequence was 81.1% and 83.2% identical to the human and bovine sequences, respectively. A 4 bp deletion was identified in diseased Persian cats; this deletion caused a frameshift from codon 583 and premature termination at codon 645. No enzyme activity could be detected in the cat liver. Pet longhair cats with a mild phenotype had 2% normal enzyme activity; this cat had no 4 bp deletion. Tollersrud et al. (Eur J Biochem 246: 410-419. 1997) (Non-Patent Document 17) purified bovine kidney enzyme to homogeneity and cloned its gene. This gene was composed of 24 exons spanning 16 kb. Based on its gene sequence, the authors identified two mutations in cattle.

Medical needs for the treatment of α-mannosidosis Fully recognized that there is a lack of effective treatment for α-mannosidosis in view of the severity of clinical signs resulting from the accumulation of high mannose oligosaccharides Has been. At present, the primary therapeutic option in the treatment of this disease is bone marrow transplantation, but it is an object of the present invention to promote enzyme replacement therapy as a candidate for future alternatives.

Bone marrow transplantation
In 1996, Walkley et al. (Proc. Nat. Acad. Sci. 91: 2970-2974, 1994) reported that three kittens with mannosidosis were bone marrow transplanted (BMT) in 1991. ) Was published. In two sacrificed animals, normalization was observed not only in the body, but more importantly in the brain. The third cat was good after 6 years. Usually, untreated cats die within 3-6 months. In 1987, a child with mannosidosis was treated with BMT (Will et al. Arch Dis Child 1987 Oct; 62 (10): 1044-9 (Non-patent Document 18)). He died 18 weeks later due to surgical complications. Only a small amount of enzyme activity was found in the brain. This unfortunate result can be explained by a strong immunosuppressive treatment before death, or by the time it takes for enzyme activity to increase in the brain after BMT. The donor was a mother (which should have been expected to have less than 50% enzyme activity as a carrier), or BMT in humans has an effect on brain enzyme function Maybe not. Although results vary, several bone marrow transplant efforts have shown that successful engraftment can at least partially correct the clinical signs of α-mannosidosis. However, attempts to reduce serious surgical complications when applying bone marrow transplantation as a treatment for humans have not been successful.

Enzyme replacement therapy When lysosomal storage disease was discovered, it was expected that this could be treated by enzyme replacement. Enzyme replacement therapy has proven effective in Gaucher disease. When exogenous lysosomal glucocerebrosidase is injected into a patient, the enzyme is taken up by enzyme-deficient cells (Barton et al. N Engl J Med 324: 1464-1470). Such uptake is due to specific receptors on the cell surface, such as mannose-6-phosphate receptors that are almost ubiquitous on the surface of the cell and other receptors, such as cells of the monocyte / macrophage cell line and hepatocytes Are regulated by asialoglycoprotein receptors and mannose receptors which are restricted to specific cell types such as The cellular uptake of this enzyme is therefore highly dependent on its glycosylation profile. When properly designed, this defective enzyme can be replaced by regular injections of exogenous enzymes in the same manner that insulin is administered to diabetic patients. In vitro studies in which purified active lysosomal α-mannosidase was added to the medium of enzyme-deficient fibroblasts showed correction of lysosomal substrate accumulation. On the other hand, in vivo treatment has been hampered, in part, by problems of producing sufficient amounts of enzyme due to difficulties in large-scale production and purification procedures, and complications resulting from immune responses to exogenous enzymes.

  More importantly, however, special consideration is given to lysosomal storage diseases associated with major neurological elements, such as α-mannosidosis, where clinical signs are associated with increased lysosomal accumulation in the central nervous system. Is directed. This is because enzyme replacement therapy has not proven to be effective against acute neuropathic variants of Gaucher disease (Prows et al. Am J Med Genet 71: 16-21) Reference 20)).

  Delivery of therapeutic enzymes to the brain is hampered by the fact that these large molecules are not transported across the blood brain barrier. From the general knowledge that the blood-brain barrier must be avoided to obtain therapeutic effects in the brain, the use of a wide variety of delivery systems has been investigated. These include invasive techniques such as osmotic opening of the blood brain barrier using, for example, mannitol, and non-invasive techniques such as endocytosis via receptors for chimeric enzymes. The use of invasive techniques should be avoided because the enzyme replacement method may require periodic administration of the enzyme. The use of non-invasive techniques has only recently provided promising results in animal models (see below for α-mannosidosis, for other lysosomal disorders, see eg Grubb et al. PNAS 2008 , 105 (7) pp.2616-2621 (see Non-Patent Document 21)). It is believed that reduced accumulation in visceral organs and meninges can reduce the amount of oligosaccharides delivered to the brain. However, this idea does not appear to be applicable to lysosomal disorders where neurological damage is central and severe (Neufeld, EF Enzyme replacement therapy, in "Lysosomal disorders of the brain" ( Platt, FM Walkley, S. V: eds Oxford University Press) (Non-Patent Document 22).

  However, Roces et al. Human Molecular Genetics 2004, 13 (18) pp. 1979-1988 (Non-Patent Document 23), Blanza et al. Human Molecular Genetics 2008, 17 (22) pp. 3437-3445 (Non-Patent Document 24) ) And WO 05/094874, increase LAMAN levels in the central nervous system of animals using, for example, intravenous injection of formulations containing α-mannosidase, thereby It has been shown that it is possible to reduce intracellular levels of mannose-rich neutral oligosaccharides in one or more regions of the central nervous system. This indicates that recombinant α-mannosidase is useful in enzyme replacement therapy for patients suffering from α-mannosidosis. Thus, one major hurdle left to provide effective treatment of α-mannosidosis using enzyme replacement methods provides a sufficient amount of pure recombinant α-mannosidase in a cost-effective manner. That is.

Production and purification of α-mannosidase
WO 02/099092 (Patent Document 2) discloses a small-scale production method of rhLAMAN in CHO cells using a serum-free medium at 37 ° C. Numerous chromatographic purification steps, including crude enzyme diafiltration and weak anion exchange chromatography using DEAE Sepharose FF columns followed by hydrophobic interaction chromatography and mixed-mode chromatography A small-scale purification method is also described.

  WO 05/094874 (Patent Document 1) discloses a small-scale production method of rhLAMAN in Chinese hamster ovary (CHO) cells using a serum-free medium at 37 ° C. A small-scale purification method similar to that of WO 02/099092 (Patent Document 2) is also described.

  WO 05/077093 (Patent Document 3) describes the production of a highly phosphorylated lysozyme enzyme. Example IV describes a method for purifying acid α-glucosidase (GAA) using a multimode resin (Blue-Sepharose). GAA is a lysozyme enzyme but differs greatly from rhLAMAN. GAA is highly phosphorylated, whereas rhLAMAN is less phosphorylated. Furthermore, the sequence identity score is less than 12% between GAA and rhLAMAN, and finally their theoretical isoelectric points differ by more than 1 pH unit (5.42 and 6.48, respectively). Therefore, the GAA purification method described in WO 05/077093 (Patent Document 3) is not applicable to rhLAMAN.

  A small-scale production method of rhLAMAN in CHO cells using 0.25% (V / V) serum and DMSO addition is disclosed (Berg et al. Molecular Genetics and Metabolism, 73, pp 18-29, 2001 (Non-Patent Document 4). )). The document also describes two purification methods including a) a three-step procedure including ultrafiltration, anion exchange chromatography and gel filtration or b) a one-step immunoaffinity chromatography. The literature also describes a) how to fully fragment a 130 kDa enzyme into 55 kDa and 72 kDa fragments, and b) partial fragmentation from a 130 kDa precursor to substantial 55 and 72 kDa fragments. The law is also disclosed.

  Therefore, improved methods for the production and purification of recombinant α-mannosidase would be beneficial. In particular, improved methods for large-scale culture of cell lines capable of expressing α-mannosidase and more efficient large-scale purification for isolating pure α-mannosidase with high enzymatic activity from cell culture The method will be beneficial.

WO 05/094874 WO 02/099092 WO 05/077093

Hirsch et al. EMBO J. 22, 1036-1046, 2003 Saint-Pol et al. J. Biol. Chem. 274, 13547-13555, 1999 Aronson and Kuranda FASEB J 3: 2615-2622. 1989 Berg et al. Molecular Genetics and Metabolism, 73, pp 18-29, 2001 Nilssen et al. Hum. Mol. Genet. 6, 717-726. 1997 Kaneda et al. Chromosoma 95: 8-12. 1987 Riise et al. Genomics 42: 200-207, 1997 Liao et al. J. Biol. Chem. 271, 28348-28358. 1996 Nebes et al. Biochem. Biophys. Res. Commun. 200, 239-245. 1994 Chester et al., In: Durand P, O'Brian J (eds) Genetic errors of glycoprotein metabolism. Edi-Ermes, Milan, pp 89-120. 1982 Thomas: Beaudet. In: Scriver CR, Beaudet AL, Sly WA, Valle D (eds) The metabolic and molecular bases of inherited disease.Vol 5. McGraw-Hill, New York, pp 2529-2562. 1995 Hocking et al. Biochem J 128: 69-78. 1972 Walkley et al. Proc. Nat. Acad. Sci. 91: 2970-2974, 1994 Crawley et al. Pediatr Res 46: 501-509, 1999 Stinchi et al. Hum Mol Genet 8: 1366-72, 1999 Berg et al. (Biochem J. 328: 863-870. 1997) Tollersrud et al. (Eur J Biochem 246: 410-419. 1997) Will et al. Arch Dis Child 1987 Oct; 62 (10): 1044-9 Barton et al. N Engl J Med 324: 1464-1470 Prows et al. Am J Med Genet 71: 16-21 Grubb et al. PNAS 2008, 105 (7) pp.2616-2621 Neufeld, E. F. Enzyme replacement therapy, in "Lysosomal disorders of the brain" (Platt, F. M. Walkley, S. V: eds Oxford University Press) Roces et al. Human Molecular Genetics 2004, 13 (18) pp. 1979-1988 Blanz et al. Human Molecular Genetics 2008, 17 (22) pp. 3437-3445

  Accordingly, the object of the present invention relates to a method for producing and purifying recombinant α-mannosidase.

  In particular, it provides a scalable and production method and purification method that solves the problems of the prior art described above by providing a sufficient amount of high purity α-mannosidase with high enzymatic activity, thereby reducing α-mannosidosis It is an object of the present invention to provide treatment for affected patients.

  Accordingly, one aspect of the present invention is a method for purifying recombinant α-mannosidase from cell culture, wherein the fraction of the cell culture containing recombinant α-mannosidase is subjected to chromatography on a resin containing a multimodal ligand. Related to the method. The inventors have surprisingly obtained by this purification method a composition comprising recombinant α-mannosidase having the desired 130 kDa glycoprotein species in higher purity and higher ratio than previously achieved. I discovered that. Achieving a consistently high unfragmented 130 kDa glycoprotein ratio (eg, greater than 80%) after purification provides a more uniform product compared to the fragmenting enzyme, which in turn turns to pharmaceutical grade This is beneficial in terms of improving the ability to obtain the product.

Another aspect of the present invention is a method for semi-batch production or continuous production of recombinant α-mannosidase, comprising the following steps:
a. On day 0, inoculating cells capable of producing recombinant α-mannosidase into a production reactor containing a base medium and supplying a cell culture;
b. adding a feed medium to the cell culture at least once from day 1;
c. After day 3 or when viable cell density exceeds 2.1 MVC / mL, whichever comes first, the temperature of the cell culture is at most 35 ° C., for example 34 ° C., 33 ° C., 32 ° C., preferably Process to adjust to 31 degrees Celsius at the maximum,
Relates to a method comprising:

  The inventors have surprisingly obtained a cell culture containing recombinant α-mannosidase in high yield by the above production method, which can be immediately transferred to the purification column of the present invention without dilution. I found it possible.

  Yet another aspect of the present invention is to provide a composition comprising purified recombinant α-mannosidase, wherein at least 80% of the α-mannosidase is present as a 130 kDa glycoprotein.

  One other aspect of the invention is a composition comprising purified recombinant α-mannosidase for use in the treatment of α-mannosidosis.

であり、ここで、式（II）および（III）の物質のRは式（IV）の官能基：

である、本発明1001〜1003のいずれかの方法。
[本発明1005]
マルチモードリガンドを含む樹脂にロードされた細胞培養物のフラクションが、イソプロパノール、好ましくは少なくとも1 % (V:V)のイソプロパノール、例えば少なくとも2 %、3 %、4 %、4.5 % (V:V)のイソプロパノール、好ましくは少なくとも5 % (V:V)のイソプロパノールを含む溶液を用いる少なくとも1回の洗浄工程に供される、本発明1001〜1004のいずれかの方法。
[本発明1006]
組み換えα-マンノシダーゼを含む第1の溶出液が、エチレングリコールまたはプロピレングリコールを含む水溶液を用いてマルチモードリガンドを含む樹脂から溶出される、本発明1001〜1005のいずれかの方法。
[本発明1007]
マルチモードリガンドを含む樹脂から得られるα-マンノシダーゼを含む第1の溶出液が、
(i) α-マンノシダーゼを含むフラクションを疎水性相互作用クロマトグラフィー樹脂に適用して、組み換えα-マンノシダーゼを含む溶出液を供給する工程、
(ii) α-マンノシダーゼを含むフラクションをミックスモードイオン交換樹脂に通して混入物質を保持させ、組み換えα-マンノシダーゼを含むフロースルー液を供給する工程、および
(iii) α-マンノシダーゼを含むフラクションを陰イオン交換樹脂によるクロマトグラフィーに供して、組み換えα-マンノシダーゼを含む溶出液を供給する工程
を含む方法にさらに供される、本発明1001〜1006のいずれかの方法。
[本発明1008]
α-マンノシダーゼが、
(A) SEQ ID NO 2で示される配列
(B) (A)の配列のアナログ
(C) (A)または(B)の配列の部分配列
から選択される配列を有する、本発明1001〜1007のいずれかの方法。
[本発明1009]
本発明1001〜1008のいずれかの精製方法により得ることができるα-マンノシダーゼを含む組成物。
[本発明1010]
a. 第0日に、組み換えα-マンノシダーゼを産生することができる細胞を、ベース培地を含む生産用リアクターに接種し、細胞培養物を供給する工程、
b. 第1日から少なくとも1度、細胞培養物にフィード培地を添加する工程、
c. 第3日以降または生存細胞密度が2.1 MVC/mLを上回ったときのいずれか早い方において、細胞培養物の温度を、最高でも35℃、例えば34℃、33℃、32℃、好ましくは最高でも31℃に調節する工程
を含む、組み換えα-マンノシダーゼの半回分生産または連続生産のための方法。
[本発明1011]
d. 本発明1001〜1008のいずれかの精製方法
をさらに含む、本発明1010の方法。
[本発明1012]
細胞培養物が、動物由来の任意の補助成分、例えばタラ肝油補助成分を本質的に含まない、本発明1010または1011の方法。
[本発明1013]
少なくとも30 L、例えば少なくとも50 L、75 L、100 L、150 L、200 L、好ましくは少なくとも250 Lの容量で実施される、本発明1010〜1012のいずれかの方法。
[本発明1014]
α-マンノシダーゼが、
(A) SEQ ID NO 2で示される配列
(B) (A)の配列のアナログ
(C) (A)または(B)の配列の部分配列
から選択される配列を有する、本発明1010〜1013のいずれかの方法。
[本発明1015]
本発明1010〜1014のいずれかの生産方法により得ることができる、α-マンノシダーゼを含む組成物。
[本発明1016]
精製された組み換えα-マンノシダーゼを含む組成物であって、該α-マンノシダーゼの少なくとも80 %が130 kDa糖タンパク質として存在する、組成物。
[本発明1017]
組み換えα-マンノシダーゼが、+5℃で保存される場合は少なくとも4日間、または-20℃で保存される場合は少なくとも24ヶ月間、溶液中で安定性を維持する、本発明1016の組成物。
[本発明1018]
α-マンノシダーゼが、
(A) SEQ ID NO 2で示される配列
(B) (A)の配列のアナログ
(C) (A)または(B)の配列の部分配列
から選択される配列を有する、本発明1016または1017の組成物。
[本発明1019]
医薬として使用するための、本発明1009および1015〜1018のいずれかの組成物。
[本発明1020]
α-マンノシドーシスの処置において使用するための、本発明1009および1015〜1018のいずれかの組成物。
[本発明1021]
α-マンノシドーシス処置用の医薬の調製のための、本発明1009および1015〜1018のいずれかの組成物の使用。
[本発明1022]
α-マンノシドーシスを処置する、および／または、α-マンノシドーシスに関連する症状を低減もしくは軽減する方法であって、本発明1009および1015〜1018のいずれかの精製された組み換えα-マンノシダーゼを含む組成物を、それを必要とする対象に投与する工程を含む、方法。
Yet another aspect of the present invention is a method for treating α-mannosidosis and / or reducing or alleviating symptoms associated with α-mannosidosis, comprising a composition comprising purified recombinant α-mannosidase A method comprising administering to a subject in need thereof.
[Invention 1001]
A method for purifying recombinant α-mannosidase from cell culture, wherein a fraction of the cell culture containing recombinant α-mannosidase is subjected to chromatography on a resin containing a multimodal ligand.
[Invention 1002]
The method of the invention 1001, wherein the fraction of the cell culture comprising recombinant α-mannosidase is a clarified undiluted harvest.
[Invention 1003]
The method of the present invention 1001 or 1002, wherein the multimodal ligand bonded to the resin is a substance having a carboxylic acid group or a sulfonic acid group.
[Invention 1004]
The multimodal ligand bound to the resin is a substance of formula (I), (II) or (III):

Where R of the substances of formula (II) and (III) is a functional group of formula (IV):

The method according to any one of 1001 to 1003 of the present invention.
[Invention 1005]
The fraction of the cell culture loaded on the resin containing the multimodal ligand is isopropanol, preferably at least 1% (V: V) isopropanol, for example at least 2%, 3%, 4%, 4.5% (V: V) A process according to any of the inventions 1001 to 1004, which is subjected to at least one washing step using a solution comprising isopropanol, preferably at least 5% (V: V) isopropanol.
[Invention 1006]
The method according to any of claims 1001 to 1005, wherein the first eluate comprising recombinant α-mannosidase is eluted from the resin comprising the multimodal ligand using an aqueous solution comprising ethylene glycol or propylene glycol.
[Invention 1007]
A first eluate containing α-mannosidase obtained from a resin containing a multimodal ligand is
(i) applying a fraction containing α-mannosidase to a hydrophobic interaction chromatography resin to supply an eluate containing recombinant α-mannosidase;
(ii) passing a fraction containing α-mannosidase through a mixed mode ion exchange resin to retain contaminants and supplying a flow-through solution containing recombinant α-mannosidase; and
(iii) A step of subjecting a fraction containing α-mannosidase to chromatography using an anion exchange resin to supply an eluate containing recombinant α-mannosidase
The method according to any one of the inventions 1001 to 1006, further provided for a method comprising
[Invention 1008]
α-mannosidase is
(A) Sequence indicated by SEQ ID NO 2
(B) Analog of sequence (A)
(C) Partial sequence of sequence (A) or (B)
The method of any one of the inventions 1001 to 1007, having a sequence selected from:
[Invention 1009]
A composition comprising α-mannosidase obtainable by the purification method of any one of the present inventions 1001 to 1008.
[Invention 1010]
a. On day 0, inoculating cells capable of producing recombinant α-mannosidase into a production reactor containing a base medium and supplying a cell culture;
b. adding a feed medium to the cell culture at least once from day 1;
c. After day 3 or when viable cell density exceeds 2.1 MVC / mL, whichever comes first, the temperature of the cell culture is at most 35 ° C., for example 34 ° C., 33 ° C., 32 ° C., preferably Process to adjust to 31 ℃ at the maximum
A process for semi-batch or continuous production of recombinant α-mannosidase, comprising:
[Invention 1011]
d. Purification method according to any one of the inventions 1001 to 1008
The method of the present invention 1010 further comprising:
[Invention 1012]
The method of 1010 or 1011 of the present invention, wherein the cell culture is essentially free of any supplemental ingredients derived from animals, such as cod liver oil supplements.
[Invention 1013]
The method of any of the invention 1010 to 1012, carried out in a volume of at least 30 L, such as at least 50 L, 75 L, 100 L, 150 L, 200 L, preferably at least 250 L.
[Invention 1014]
α-mannosidase is
(A) Sequence indicated by SEQ ID NO 2
(B) Analog of sequence (A)
(C) Partial sequence of sequence (A) or (B)
The method of any of the inventions 1010 to 101 having a sequence selected from:
[Invention 1015]
A composition comprising α-mannosidase, which can be obtained by the production method according to any one of the present invention 1010 to 1014.
[Invention 1016]
A composition comprising purified recombinant α-mannosidase, wherein at least 80% of the α-mannosidase is present as a 130 kDa glycoprotein.
[Invention 1017]
The composition of the invention 1016 wherein the recombinant α-mannosidase remains stable in solution for at least 4 days when stored at + 5 ° C. or for at least 24 months when stored at −20 ° C.
[Invention 1018]
α-mannosidase is
(A) Sequence indicated by SEQ ID NO 2
(B) Analog of sequence (A)
(C) Partial sequence of sequence (A) or (B)
The composition of this invention 1016 or 1017 having a sequence selected from:
[Invention 1019]
The composition of any of the inventions 1009 and 1015-1018 for use as a medicament.
[Invention 1020]
The composition of any of the present invention 1009 and 1015-1018 for use in the treatment of alpha-mannosidosis.
[Invention 1021]
Use of the composition of any of the inventions 1009 and 1015-1018 for the preparation of a medicament for the treatment of α-mannosidosis.
[Invention 1022]
A method of treating α-mannosidosis and / or reducing or alleviating symptoms associated with α-mannosidosis, wherein the purified recombinant α-mannosidase of any of the present invention 1009 and 1015-1018 Administering a composition comprising: to a subject in need thereof.

An overview of the preferred purification method of the present invention designed for α-mannosidase from collection to drug substance filing is shown. An example of a Capto ™ MMC column chromatogram of α-mannosidase is shown. An example of a butyl Sepharose ™ FF column chromatogram of α-mannosidase is shown. An example of a CHT type 1 column chromatogram of α-mannosidase is shown. An example of a Q sepharose ™ HP column chromatogram of α-mannosidase is shown. Figure 2 shows an SDS-page chromatogram of a purified α-mannosidase composition showing the distribution of 130 kDa, 75 kDa and 55 kDa glycoprotein species. Three HPLC diagrams for purified α-mannosidase are shown, in which the amount of 130 kDa species is shown in comparison with 55 and 75 kDa species. The first peak from the left is the 55 kDa species, followed by each of the 130 kDa and 75 kDa species. The two-step method does not use a multi-mode ligand chromatography step, but the three-step method and the four-step method use a multi-mode ligand chromatography step.

  In the following, the present invention will be described in more detail.

Detailed Description of the Invention Definitions Prior to discussing the present invention in further detail, the following terms and conventions will first be defined.

Recombinant α-mannosidase In the context of the present invention, recombinant α-mannosidase is defined as an α-mannosidase that does not equal all or part of the wild-type α-mannosidase found in nature by its origin or manipulation. Thus, it is a recombinant technique involving a recombinant DNA molecule that is a hybrid DNA sequence that is a sequence of at least two fused DNA sequences, the first sequence of which is normally not fused to the second sequence in nature. It is constructed using The recombinant α-mannosidase protein may be of human or non-human origin. In particular, it can be a recombinant human lysosomal α-mannosidase (rhLAMAN). The α-mannosidase product can be a single polypeptide or a mixture of a single polypeptide and fractions thereof. Also, α-mannosidase is subject to post-translational modification and can therefore be in the form of a glycoprotein.

Cell culture Cell culture is a method of growing cells under controlled conditions. In the context of the present invention, the cells in the cell culture are specifically designed to express a protein of interest, such as recombinant α-mannosidase. Cell culture can be performed in bioreactors that are specially designed to achieve control of chemical and physical conditions.

Fraction In the context of the present invention, a fraction means a fraction of a cell culture. The fraction may constitute a whole cell culture, but in many cases a processed fraction of the culture, such as a clarified, filtered, concentrated, diluted or partially purified Fraction.

Resin In the context of the present invention, the resin forms the basis of a stationary phase in a chromatographic system, which can be used in a variety of ways to provide a certain amount of affinity for a particular molecule or protein of interest. A chemical group or substance is added. The resin is often a polymer bead to which a ligand is covalently attached and is insoluble in the liquid mobile phase used.

Multimodal ligand A multimodal ligand means any ligand designed to interact with a molecule or protein of interest in at least two ways. Individual interactions can be hydrophobic, hydrophilic, ionic, van der Waals interactions, hydrogen bonds, or chemical or physical interactions between any other molecules, independently. In the context of the present invention, a ligand is an organic chemical added to a resin as described above. Multimodal ligands have different affinities for different substances dissolved in the mobile phase and passing through the chromatography column. Differences in affinity result in variations in the retention times of different substances in the chromatography column, thereby realizing separation of those substances. The retention time also depends on other factors such as mobile phase components, pH and temperature. Resins comprising multimodal ligands are sometimes referred to as “mixed mode” resins, but in the context of the present invention, resins comprising multimodal ligands comprise several different “ligands” on the same resin; Ceramic hydroxyapatite resins (CHT) should not be confused with so-called “mixed mode ion exchange resins” which can have opposite charges, such as —OH, —Ca ⁺ and —PO ₄ ²⁻ .

Load In the context of the present invention, load means the transfer of a harvest, eluate or other solution to a chromatography system, for example a chromatography column containing the resin as a stationary phase.

Buffer The term buffer is well known as a general expression for a solution that resists changes in pH, including either weak acids and / or their corresponding salts or weak bases and / or their corresponding salts. In the context of the present invention, the buffers used are those suitable for use in chromatography systems, such buffers include phosphate buffers such as disodium phosphate (Na ₂ HPO ₄ ), sodium phosphate or Includes, but is not limited to, potassium phosphate, acetate buffers such as sodium acetate or potassium acetate, sulfate buffers such as sodium sulfate or potassium sulfate, ammonium sulfate or Hepes, or other buffers such as sodium borate or tris-HCl buffer. .

Ultrafiltration Ultrafiltration is a separation method that utilizes water pressure to pass molecules and solvents across a membrane containing pores of a specific size, also known as a cut-off size or cut-off value. Only molecules with a molecular weight smaller than the membrane cut-off value can pass through the membrane, and molecules with a higher molecular weight do not pass through the membrane and form a so-called retentate. Thus, molecules present in the non-permeate can be concentrated as the solvent flows out through the membrane.

  In certain embodiments, concentration of a solution or composition comprising a polypeptide, such as α-mannosidase, can be performed by tangential flow filtration (TFF). This method is particularly useful for large-scale concentration, i.e. concentration of solutions of 1 to several hundred liters in volume. This method is therefore particularly useful for the production of concentrated solutions of the polypeptide of interest on an industrial scale.

  TFF technology is based on the use of a specific device that allows the solution to be filtered to flow through a semi-permeable membrane; only molecules smaller than the membrane pores pass through the membrane to form a filtrate (non-permeate) that retains the large material to be recovered. In the TFF method, two different pressures are applied; one is to pump the solution through the system and circulate it through the system (inflow pressure), the other is a membrane on the small molecule and solvent. It is applied across the membrane to allow it to pass through (membrane pressure). The inlet pressure can typically be in the range of 1 to 3 bar, for example between 1.5 and 2 bar. The transmembrane pressure (TMP) can typically be greater than 1 bar. A concentrated composition of the polypeptide of interest can be recovered as a non-permeate when TFF is used to concentrate the composition. Membranes useful for TFF can typically be made of regenerated cellulose or polyethersulfone (PES).

Diafiltration In the context of the present invention, diafiltration leaves the species of interest in the non-permeate, i.e. does not pass through the filter, and other components such as buffers and salts pass through the filter. It is. Thus, diafiltration can be used, for example, to exchange one buffer for another buffer or to concentrate a solution containing a species of interest, such as recombinant α-mannosidase.

  A first aspect of the present invention is a method for purifying recombinant α-mannosidase from cell culture, wherein a fraction of the cell culture containing recombinant α-mannosidase is subjected to chromatography with a resin containing a multimodal ligand. To provide a method. The advantage of using resins containing multimodal ligands in the present invention is that these resins can bind to α-mannosidase species in solutions with high conductivity levels. This has the advantage that an undiluted harvest with a high conductivity level can be used and no exchange of the harvest buffer is required. Thus, the chromatography step involving the multimodal ligand may preferably be the first chromatography step after isolating the fraction from the cell culture. Chromatographic processes involving multimodal ligands often involve proteins of interest initially retained (ie, captured) on the column, whereas many impurities pass through the column during the washing step. , Sometimes referred to as “capturing step”. The protein is then eluted using a specific elution buffer.

  Accordingly, in one aspect of the invention, a method is provided wherein the fraction of cell culture containing recombinant α-mannosidase is a clarified undiluted harvest. In the context of the present invention, the term “clarified undiluted harvest” means a harvest of cell culture that is free of unlysed or solids, ie is a clear solution. This harvest may have been subjected to processing to convert it to a clear solution. Such processing can include, but is not limited to, filtration and centrifugation. Furthermore, this harvest is not significantly diluted before being subjected to a chromatography step. The harvest is therefore diluted below 10%, such as below 7%, below 5%, below 2%, below 1%, below 0.5%, such as below 0.1%. In the most preferred embodiment, the harvest is not diluted.

  In another embodiment, a method is provided wherein the clarified undiluted harvest has a conductivity of 10-20 mS / cm, such as 12-17 mS / cm, preferably 15 mS / cm. The conductivity is measured before loading the harvest into the chromatography system.

  In one embodiment, the chromatography is performed with a resin comprising a multimodal ligand having a carboxylic acid group or a sulfonic acid group. The carboxylic acids and / or sulfonic acids contained in these ligands can be in protonated or deprotonated (salt) form, depending on the conditions of the chromatography system, in particular depending on the pH of the mobile phase.

の物質であって、式（II）および（III）の物質のRが式（IV）：

の官能基である物質である方法が提供される。 In yet another embodiment, the multimodal ligand attached to the resin is of the formula (I), (II) or (III):

Wherein R of the substances of formula (II) and (III) is of formula (IV):

A method is provided that is a material that is a functional group of

  Multimodal ligands represented by functional groups of formula (IV) are commonly referred to as “Cibracon Blue 3G” and examples of commercial products represented by substances of formulas (I), (II) and (III) are: “Capto ™ MMC”, “Capto ™ Blue” and “Blue sepharose ™ fast flow”, respectively. Other useful multimode type resins include Capto ™ Adhere, MEP HyperCel ™, HEA HyperCel ™ and PPA HyperCel ™. In the context of the present invention, such resins have proven to be particularly effective for the initial purification of undiluted harvests containing recombinant α-mannosidase.

  A further aspect of the invention is that the fraction of the cell culture loaded on the resin comprising the multimodal ligand is isopropanol, preferably at least 1% (V: V) isopropanol, such as at least 2%, 3%, 4%, A method is provided that is subjected to at least one washing step using a solution comprising 4.5% (V: V) isopropanol, preferably at least 5% (V: V) isopropanol. The advantage of using a solution containing isopropanol is that it provides sufficient removal of undesired host cell proteins (HCP's), especially that it helps remove the protease responsible for proteolysis of the desired 130 kDa rhLAMAN species. is there. HCP's should be understood as proteins that are endogenous to the host cell used in the cell culture during production. Although isopropanol is preferred, other alcohols useful in this process include ethanol, n-propanol and n-butanol.

  In yet another embodiment, a method is provided wherein the pH of the solution used in the washing step is in the range of pH 3.5 to 6.5, such as pH 4.0 to 6.0, pH 4.5 to 5.5, preferably pH 4.7 to 5.0.

  In another embodiment, the solution used in the washing step is an acetate buffer, preferably in the range of 0.05 to 1.6 M, such as 0.1 to 1.5 M, 0.5 to 1.4 M, 0.7 to 1.3 M, 0.8 to 1.2 M, 0.9 to 1.1 M. A method comprising an acetate buffer, preferably at a concentration of 0.95 M is provided. The acetate buffer can preferably be selected from the group consisting of sodium acetate, potassium acetate, lithium acetate, ammonium acetate.

  Yet another embodiment provides a method wherein a first eluate comprising recombinant α-mannosidase is eluted from a resin comprising a multimodal ligand using an aqueous solution comprising ethylene glycol or propylene glycol. It has been found that the addition of ethylene glycol to the elution buffer greatly improves the yield of eluted recombinant α-mannosidase. Propylene glycol also improves yield, but ethylene glycol is preferred.

  One embodiment provides a method wherein the concentration of ethylene glycol or propylene glycol in the aqueous solution is 20-60%, 20-50%, 25-50%, 30-50%, 35-45%, such as 40%. .

  In a preferred embodiment, a method is provided wherein the aqueous solution comprising ethylene glycol or propylene glycol comprises sodium chloride. It has been found that the addition of sodium chloride to this solution greatly improves the yield by facilitating the elution of the rhLAMAN enzyme.

  In another embodiment, the concentration of sodium chloride in the aqueous solution comprising ethylene glycol or propylene glycol ranges from 0.2 to 2.4 M, such as 0.4 to 2.2 M, 0.6 to 2.0 M, 0.8 to 1.9 M, 1.0 to 1.8 M, 1.2 to The range is 1.7 M, 1.4 to 1.6 M, preferably 1.5 M. Alternatively, the concentration of sodium chloride can be in the range of 0.2-1.6 M or in the range of 1.4-2.4 M.

  In a preferred embodiment, the aqueous solution containing ethylene glycol or propylene glycol contains a buffer. The buffer may preferably be a phosphate buffer such as sodium phosphate or potassium phosphate. Although phosphate buffers are preferred, additional buffers useful for this aqueous solution include citrate and borate buffers, Tris, MES, MOPS and Hepes buffers.

  In another preferred embodiment, the concentration of the buffer salt in the aqueous solution comprising ethylene glycol or propylene glycol is 50-350 mM, 55-300 mM, 65-280 mM, 70-250 mM, 75-200, 80-200 mM. 85-150 mM, preferably 90 mM.

  In yet another preferred embodiment, the pH of the aqueous solution comprising ethylene glycol or propylene glycol is pH 7.0 to 9.0, such as pH 7.1 to 8.5, pH 7.2 to 8.3, pH 7.5 to 8.0, preferably pH 7.7.

In one embodiment, there is provided a method wherein a first eluate comprising α-mannosidase obtained from a resin comprising a multimodal ligand is further subjected to a method comprising the following steps:
(i) applying a fraction containing α-mannosidase to a hydrophobic interaction chromatography resin to supply an eluate containing recombinant α-mannosidase;
(ii) passing a fraction containing α-mannosidase through a mixed mode ion exchange resin to retain contaminants and supplying a flow-through solution containing recombinant α-mannosidase; and
(iii) A step of subjecting a fraction containing α-mannosidase to chromatography using an anion exchange resin to supply an eluate containing recombinant α-mannosidase.

  In one embodiment, the method comprises steps (i) to (iii) above, wherein the fraction of step (i) has been subjected to purification with a resin containing a multimodal ligand, and the fraction of step (ii) There is provided a method wherein the eluate of i) is derived and the fraction of step (iii) is derived from the flow-through of step (ii). In other words, steps (i) to (iii) are performed in the order listed. However, an intermediate step between steps (i) to (iii) is not excluded. These can be intermediate purification steps and / or virus reduction or removal steps.

  In a preferred embodiment, the hydrophobic interaction chromatography resin of step (i) is an alkyl substituted resin, preferably a butyl sepharose resin. Alkyl substituted resins can include ethyl, butyl and octyl sepharose resins. In addition, phenyl sepharose resins are also applicable. Examples of such resins include Butyl-S Sepharose ™ 6 Fast Flow, Butyl Sepharose ™ 4 Fast Flow, Octyl Sepharose ™ 4 Fast Flow, Phenyl Sepharose ™ 6 Fast Flow (high sub) and Phenyl Sepharose ™ 6 Fast Flow (low sub), Butyl Sepharose ™ High Performance, and Phenyl Sepharose ™ High Performance. The advantage of the purification process using hydrophobic interaction resins, especially butyl sepharose resin, is that it efficiently removes host cell proteins, especially DNA residues, while maintaining good yields of rhLAMAN enzyme.

  In yet another embodiment, step (i) comprises at least one washing step, and the solution used for washing comprises a phosphate buffer and an acetate buffer, preferably sodium phosphate and sodium acetate. This dual buffer wash process has proven to be particularly effective in removing impurities such as host cell proteins and DNA residues.

  In yet another embodiment, the phosphate buffer concentration in the dual buffer wash of step (i) is in the range of 5-40 mM, such as 10-30 mM, 15-25 mM, preferably 20 mM. The concentration ranges from 0.9 to 1.5 M, for example 1.0 to 1.4 M, 1.1 to 1.3 M, preferably 1.2 M.

  In another embodiment, step (i) comprises at least one washing step, and the solution used for washing comprises only one buffer, preferably a phosphate buffer, preferably sodium phosphate.

  In another embodiment, one buffer of at least one washing step comprising only one buffer is present at a concentration in the range 0.4-0.8 M, such as 0.5-0-7 M, preferably 0.6 M.

  In one embodiment, a method is provided wherein the mixed mode ion exchange resin of step (ii) is a ceramic hydroxyapatite or fluoroapatite resin, preferably a ceramic hydroxyapatite type I (CHT I) resin. By applying this chromatographic step, significant amounts of DNA impurities are effectively separated from the recombinant α-mannosidase composition and the host cell proteins bind but the rhLAMAN enzyme product does not bind to the column. Was shown to do.

  In another embodiment, the anion exchange resin of step (iii) is a strong anion exchange resin, such as a quaternary ammonium strong anion exchange resin. Such resins include, but are not limited to, the following examples: Q-sepharose ™ HP, Q-sepharose ™ FF, DEAE-sepharose ™, Capto ™ Q, Uno ™ ) Q, ANX sepharose (trademark).

  In yet another aspect, a method is provided wherein the virus inactivation step is preferably performed between step (ii) and step (iii).

  In a preferred embodiment, the virus inactivation step comprises a mixing of the flow-through solution of step (ii) with an aqueous solution of isopropanol for at least 2 hours (1: 1 V / V of flow-through solution / isopropanol aqueous solution), Including concentration by ultrafiltration and removal of isopropanol using diafiltration. The aqueous isopropanol solution during inactivation can be 10-50% isopropanol, for example in the range 20-40%, 25-35%, 28-32%, preferably 30% isopropanol. Thus, a 1: 1 V / V solution of flow-through and aqueous isopropanol has a final isopropanol concentration of 15%.

  Another preferred embodiment is the method wherein the virus reduction step is preferably performed after the chromatography step (iii).

  In one embodiment, the virus reduction step comprises a solution, preferably a step, comprising a recombinant α-mannosidase through a filter, preferably a virus removal filter such as an ultipor ™ VF grade DV20 filter or a planova ™ 15N or 20N filter. including filtration of the eluate of (iii). Preferably, a Planova ™ 15N filter is used.

  The purification method of the present invention is advantageous in that it can be performed on a large scale, and thus in a preferred embodiment, the method is at least 0.5 L, such as at least 1.0 L, 2.0 L, 5.0 L, 10 L, preferably at least 13.0. Performed on a chromatography column having a column volume of L.

In another aspect of the invention, α-mannosidase is
(A) Sequence indicated by SEQ ID NO 2
(B) A sequence analog
(C) The above purification method having a sequence selected from a partial sequence of the sequence of (A) or (B) is provided.

  Here, the sequence represented by SEQ ID NO 2 represents the amino acid sequence of the recombinant human lysosomal α-mannosidase (rhLAMAN) provided in WO 02/099092.

A `` partial sequence '' is a parent that has a size of 50% or more of the parent sequence, e.g. 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more or 95% or more of the parent sequence. Means a fragment of a sequence. Thus, the partial sequence of interest is 505-1009 contiguous amino acid residues, e.g., 525-1009, 550-1009, 575-1009, 600-1009, 625-1009, 650-1009, 675-1009, 700-1009. 725-1009, 750-1009, 775-1009, 800-1009, 825-10009, 850-1009, 875-1009, 900-1009, 925-1009, 950-1009, 975-1009, 980-1009, 990 It may have a length of ˜1009 or such as 1000-1009 contiguous amino acid residues. Furthermore, the relevant partial sequence of SEQ ID NO: 2 or an analog thereof must retain a catalytic site. Although the 3D structure of human LAMAN is not known, the 3D structure of bovine LAMAN has been reported, and based on the data, the following amino acids are also involved in the active site and / or required for activity in human LAMAN It is concluded that it is responsible for the coordination of Zn ²⁺ atoms: AA 72 = H, AA 74 = D, AA 196 = D, AA 446 = H (UniProtKB / Swiss-Prot database: O00754, MA2B1_HUMAN_, Heikinheimo et al. J. Mol. Biol. 327, 631-644, 2003). Mutations of AA 72 and 196 in human LAMAN have been shown to almost completely abolish enzyme activity (Hansen et al., Biochem. J. (2004), 381, pp. 537-567). In order to exert the activity, the rhLAMAN partial sequence preferably has a region containing at least the above-mentioned four amino acids.

  Preferably, the rhLAMAN subsequence also includes one or more additional conformational moieties including, for example, binding sites, β-turns, disulfide bridges, stop codons, and the like. There are several disease-induced mutations in the human form of LAMAN, which are specific amino acids such as AA 53, 72, 77, 188, 200, 355, 356, 359, 402, 453, 461, 518, 563, 639. 714, 750, 760, 801, 809, 916 (human gene mutation database, HMDG® professional, Cardiff University, 2009) and AA 133, 310, 367, 497, 645, There are also amino acids important for glycosylation, including 651, 692, 766, 832, 930 and 989, and amino acids involved in disulfide bridges such as AA 55 + 358, 268 + 273, 412 + 472 and 493 + 501.

  “Analog” means a sequence having a certain percentage of sequence identity to the parent sequence, which is at least 60% sequence identity, eg, at least 70%, 80%, 85%, 90%, 95%, There may be 98% or preferably 99% sequence identity. The analog and the partial sequence described above are preferably functionally equivalent to α-mannosidase having the amino acid sequence shown in SEQ ID NO: 2 in the sense that they can exert substantially the same enzymatic activity. Will be understood.

  The term “substantially the same enzyme activity” means at least 50% of the activity of the natural enzyme, preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably Means an equivalent moiety or analog having at least 85%, more preferably at least 90%, more preferably at least 95% and more preferably at least 97%, at least 98% or at least 99%. An example of an analog that is functionally equivalent to the enzyme can be a fusion protein containing the catalytic site of the enzyme in a functional form, but it can also be a homologous variant of an enzyme from another species. A fully synthetic molecule that mimics the specific enzyme activity of a related enzyme can also be a “functionally equivalent analog”. Non-human analogs of LAMAN are generally not applicable to therapy because they can induce antibody formation and cause disease in patients. However, human analogs can be useful in enzyme replacement therapy if the mutation is not disease-inducing and does not significantly reduce the desired enzyme activity. Examples of such mutations are His70Leu, Gln240Arg, Ser250Ala, Leu278Val, Ser282Pro, Thr312Ile, Ala337Arg, Ser413Asn, Ser481Ala, Gln582Glu, Arg741Gly, Thr873Pro (source: http: //www.ensemST. ). In addition, Pro669Leu and Asp402Lys have been confirmed by the present inventors not to cause disease.

In general, one of ordinary skill in the art would readily be able to construct an appropriate assay for determination of enzyme activity. For LAMAN, a suitable enzyme activity assay is disclosed in WO 02/099092, page 26, lines 8 to 28. Briefly, the following procedure can be performed using a flat bottom 96-well plate for screening purposes: 75 μl of 4X assay buffer (8 mM p-nitrophenyl-α-D-mannopyranoside, 2 mg / mL BSA , 0.4 M Na acetate (pH 4.5)) is added to 75 μl of sample or its appropriate dilution (in 10 mM Tris pH 7.4 containing 150 mM NaCl + 10% superblock). The plate is incubated for 30 minutes at 37 ° C., stopped with 75 μl 1.8 M Na ₂ CO ₃ and the absorbance at 405 nm is recorded in a plate reader. Read 96-well plate in spectrophotometer. Specific activity is defined as μmol of hydrolyzed p-nitrophenyl-α-D-mannopyranoside per mg protein per minute.

  As with the general definition, “identity” is defined herein as sequence identity at the nucleotide or amino acid level between genes or proteins, respectively. Thus, in the context of the present invention, “sequence identity” is a measure of identity at the amino acid level between proteins and a measure of identity at the nucleotide level between nucleic acids. The identity of protein sequences can be determined by comparing the amino acid sequences at predetermined positions in each sequence when the sequences are aligned. Similarly, the identity of nucleic acid sequences can be determined by comparing the nucleotide sequence at a given position in each sequence when the sequences are aligned.

  To determine the percent identity of two amino acid sequences or two nucleic acids, the alignments of those sequences are made for optimal comparison purposes (eg, for optimal alignment with a second amino acid or nucleic acid sequence). A gap may be inserted in the sequence of the first amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by those sequences (ie,% identity = # of identical positions / total of positions # (eg superimposed positions) x 100) . In one embodiment, the two sequences are the same length.

  Sequence alignment may be performed manually and the number of identical amino acids may be counted. Alternatively, the alignment of the two sequences to determine percent identity may be made using a mathematical algorithm. Such algorithms are incorporated into the NBLAST and XBLAST programs. To obtain nucleotide sequences homologous to the nucleic acid molecules of the invention, a BLAST nucleotide search can be performed under the NBLAST program, score = 100, word length = 12. In order to obtain amino acid sequences homologous to the protein molecules of the invention, a BLAST protein search can be performed under the XBLAST program, score = 50, word length = 3. Gapped BLAST can be used to perform an alignment with a gap for comparison purposes. Alternatively, iterative searches can be performed using PSI-Blast that detects distant relationships between molecules. When utilizing NBLAST, XBLAST, and Gapped BLAST programs, the default parameters of each program can be used. See http://www.ncbi.nlm.nih.gov. Alternatively, sequence identity can be calculated, for example, after alignment of sequences with the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST). In general, default settings, eg, default settings for “scoring matrix” and “gap penalty” may be used for alignment. In the context of the present invention, BLASTN and PSI BLAST default settings may be useful.

  The percent identity between two sequences can be determined using techniques similar to those described above, with or without gaps. Only exact matches are considered in calculating percent identity.

  Another aspect of the present invention is a composition comprising α-mannosidase obtainable by the purification method described above.

In a second aspect of the present invention, there is provided a semi-batch production method or a continuous production method of recombinant α-mannosidase comprising the following steps:
a. On day 0, inoculating cells capable of producing recombinant α-mannosidase into a production reactor containing a base medium and supplying a cell culture;
b. adding a feed medium to the cell culture at least once from day 1;
c. After day 3 or when viable cell density exceeds 2.1 MVC / mL, whichever comes first, the temperature of the cell culture is at most 35 ° C., for example 34 ° C., 33 ° C., 32 ° C., preferably The process of adjusting to 31 ° C at the maximum.

  In the above method, the inoculation date is defined as the 0th day, and the next day is the 1st day. The starting temperature used from day 0 to the adjustment described in section c is in the range of 36-37 ° C, preferably 36.5 ° C. It should be understood that the above temperatures are measured temperatures and not set points, ie, in the bioreactor settings used in the present invention, the above 31 ° C. temperature requires a 32 ° C. temperature set point. . Similarly, a temperature of 36.5 ° C requires a set temperature point of 37 ° C.

  Suitable host cells for the expression and production of recombinant α-mannosidase are obtained from multicellular organisms, preferably mammals. In particular, the cells used to produce recombinant α-mannosidase include monkey kidney CVI line transformed with SV40 (COS-7); human fetal kidney line (293 cells or subcloned for suspension culture growth) Baby hamster kidney cells (BHK); Chinese hamster ovary cells / -DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CVI); African green monkey kidney cells (VERO-76); human cervix Cancer cells (HELA); Canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); Human lung cells (W138); Human liver cells (Hep G2, HB 8065); Mouse breast cancer (MMT 060562); TRI cells; It can be selected from the group consisting of MRC 5 cells; FS4 cells; and the human liver cancer line (Hep G2). Insect cell lines or human fibroblasts are also available as suitable host cells.

Production of recombinant α-mannosidase is performed using cells transfected with an appropriate nucleic acid construct using techniques known to those skilled in the art. In particular, this nucleic acid construct is
(i) a nucleic acid sequence represented by SEQ ID NO: 1, and
(ii) a nucleic acid sequence encoding a partial sequence or analog of the sequence represented by SEQ ID NO 2 above,
A nucleic acid sequence selected from the group consisting of:

  The cell may preferably be an rLAMAN Chinese hamster ovary (CHO) cell line specially constructed for the purpose of producing a recombinant enzyme, as described in WO 02/099092. This cell line culture, DSM ACC2549, was deposited on June 6, 2002 at DSMZ GmbH, Maschroderweg 1b, D-38124, Braunschweig, Germany for the purpose of depositing patents under the Budapest Treaty. This cell can also be obtained using the expression plasmid pLamanExp1 having the sequence shown in SEQ ID NO 1.

The method of steps a to c further includes the following steps:
d. A method for purifying recombinant α-mannosidase from cell culture, wherein a fraction of the cell culture containing recombinant α-mannosidase is chromatographed on a resin containing a multimodal ligand having a carboxylic acid group or a sulfonic acid group. To provide the method,
Can be included.

  In yet another aspect, the cell culture used in the production method is essentially free of any animal-derived supplemental components, such as cod liver oil supplemental components. By avoiding the use of such auxiliary ingredients, the risk of virus contamination in the final enzyme product is reduced.

  In one preferred embodiment of the production method, the undiluted harvest for semi-batch or continuous production is at least 0.1 g / L, such as at least 0.2 g / L, 0.3 g / L, 0.4 g / L, preferably at least 0.5 g. It has an α-mannosidase concentration of / L.

  In another embodiment, the semi-batch or continuous production undiluted harvest is in the range of 3 to 35 U / mL, such as 5 to 35 U / mL, 7 to 35 U / mL, preferably 10 to 35 U / mL. Enzyme activity in the range of. It should be understood that with further method optimization, the enzyme activity of the harvest can be even higher than 35 U / mL.

  This production method is advantageous in that it can be implemented on a large scale. Thus, in one embodiment, the method for semi-batch production or continuous production is performed in a volume of at least 30 L, such as at least 50 L, 75 L, 100 L, 150 L, 200 L, preferably at least 250 L. .

In another aspect of the invention, α-mannosidase is
(A) Sequence indicated by SEQ ID NO 2
(B) A sequence analog
(C) The above production method having a sequence selected from a partial sequence of the sequence of (A) or (B) is provided.

  Another embodiment of the present invention is a composition comprising α-mannosidase obtainable by the production method described above. Such compositions may in preferred embodiments include additional active product ingredients (API), adjuvants and / or excipients.

  In a third aspect of the invention, there is provided a composition comprising purified recombinant α-mannosidase, wherein at least 80% of α-mannosidase is present as a 130 kDa glycoprotein.

  In a preferred embodiment, a composition comprising purified recombinant α-mannosidase, wherein the recombinant α-mannosidase is at least 4 days when stored at + 5 ° C. or at least 24 when stored at −20 ° C. Compositions are provided that remain stable in solution for months.

Preferred compositions of the present invention for formulation buffer solutions for rhLAMAN enzyme products are described below and the stability achieved thereby is also listed.
Na ₂ HPO ₄ 3.50 mM (dibasic sodium phosphate)
NaH ₂ PO ₄ 0.17 mM (monobasic sodium phosphate)
Glycine 27 mM
Mannitol 250 mM
pH 7.70, 290 mOsm / kg (isotonic solution)

Safety in use: Stable solution + 5 ° C-4 days
+ 20 ℃-6 hours
-20 ℃-24 months
Freeze drying + 5 ℃-24 months

In another aspect of the invention, α-mannosidase is
(A) Sequence indicated by SEQ ID NO 2
(B) A sequence analog
(C) The above composition having a sequence selected from a partial sequence of the sequence of (A) or (B) is provided.

  Another preferred embodiment is a composition comprising the purified recombinant α-mannosidase described above for use as a medicament.

  In a further embodiment, the composition comprising the purified recombinant α-mannosidase is for use in the treatment of α-mannosidosis.

  Yet another aspect is the use of a composition comprising the purified recombinant α-mannosidase described above for the preparation of a medicament for the treatment of α-mannosidosis.

  Another aspect is a method of treating α-mannosidosis and / or reducing or alleviating symptoms associated with α-mannosidosis, comprising a composition comprising the purified recombinant α-mannosidase as described above. A method comprising administering to a subject in need thereof.

  It should be noted that embodiments and features described in one context of aspects of the invention apply to other aspects of the invention.

  All patent and non-patent documents cited in this application are hereby incorporated by reference in their entirety.

  The invention will now be described in further detail by the following non-limiting examples.

Abbreviations used:
CIP: Clean In Place
CV: Column capacity
Cv: Viable cell density
DF: Diafiltration
DO: dissolved oxygen
IPA: Isopropanol
MVC / mL: 10 ⁶ viable cells / mL
NaPi: Sodium phosphate
NaAc: Sodium acetate
OD: Optical density
EG: Ethylene glycol
TFF: Tangential flow filtration
TMP: Transmembrane pressure
UF: Ultrafiltration

Example 1-Preferred Purification Procedure of the Invention General The purification procedure of the present invention for obtaining α-mannosidase in optimal yield and purity is described below. Standard resin regeneration and cleaning conditions defined for individual resins are used (see Examples 2-5). The resins used are available from GE healthcare life sciences and BioRad.

  • Supply a fraction of the harvest from the α-mannosidase product and clarify this fraction without significant dilution. Preferably no dilution is performed.

  -The above-mentioned fraction is subjected to a capture step including column chromatography using a resin containing a multimode ligand. These resins include Capto ™ MMC, Capto ™ Adhere, PlasmidSelect ™ Xtra, Capto ™ Blue, Blue Sepharose ™ Fast Flow resin, MEP HyperCel ™, HEA HyperCel ™ and Select from the group consisting of PPA HyperCel ™.

  Wash several times at pH 4.5-8.5, and wash the buffer with sodium acetate, potassium acetate, ammonium acetate, sodium phosphate, potassium phosphate, sodium sulfate, potassium sulfate, ammonium sulfate, MES, MOPS, Hepes, boric acid It is selected from the group consisting of sodium, tris-HCl, citrate buffer or a combination thereof (hereinafter buffer group A). However, in at least one wash, the wash solution contains isopropanol and the pH is between pH 4-6. The elution buffer is selected from buffer group A, and the elution solution contains ethylene glycol. The elution pH is maintained at pH 7.0-8.5.

  • For compositions containing α-mannosidase, perform an active intermediate step including column chromatography with a hydrophobic interaction resin. These resins include Butyl-S Sepharose ™ 6 Fast Flow, Butyl Sepharose ™ 4 Fast Flow, Octyl Sepharose ™ 4 Fast Flow, Phenyl Sepharose ™ 6 Fast Flow (high sub) and Phenyl Sepharose ( Trademark) 6 Fast Flow (low sub), Butyl Sepharose (trademark) High Performance, and Phenyl Sepharose (trademark) High Performance.

  Perform several washes at pH 7-8. Wash buffer is selected from buffer group A. The elution buffer is also selected from buffer group A, and the elution pH is between pH 7-8.

  For the composition containing α-mannosidase, a passive intermediate step including column chromatography with a mixed mode ion exchange resin is performed, and a flow-through solution is supplied. These resins are selected from the group consisting of ceramic hydroxyapatite type I or type II (preferably type I) resin or fluoroapatite. Wash once at pH 7-8 and supply flow-through. Wash buffer is selected from buffer group A.

  • Perform a polishing step including column chromatography with anion exchange resin on the composition containing α-mannosidase. These resins are selected from the group consisting of Q-sepharose (TM) HP, Q-sepharose (TM) FF, DEAE-sepharose (TM), Capto (TM) Q, Uno (TM) Q, ANX sepharose (TM) To do.

  Perform several washes at pH 7-8. Wash buffer is selected from buffer group A and TRIS-HCl buffer. The elution buffer is also selected from buffer group A and the elution pH is maintained between pH 7-8.

  Perform the virus inactivation step by contacting the α-mannosidase solution with isopropanol.

  -Perform a virus removal process using nanofiltration.

The yield of the purified composition containing α-mannosidase according to the present invention and the component ratio of the product are shown in Table 1 below together with a comparison with the conventional method. In the same table:
Method 1 is a preferred method according to the present invention.
Method 2 is similar to Method 1 but without a refinement step. The method does not have a wash step with isopropanol for the capture step, performs several washes in the active intermediate step, and finally no virus inactivation / removal step.
Method 3 is the one described in WO 02/099092 (no multimode ligand is used).

実施例10および図7も参照のこと。 TABLE 1 Yields and purity obtained by conventional and inventive purification procedures

See also Example 10 and FIG.

Example 2-Chromatographic Capture Step with Multimodal Ligand Clarified undiluted harvest containing α-mannosidase is used in mixed mode interaction, multimodal ligand type resin, eg in this example Bind to Capto ™ MMC. The product is eluted by increasing the salt and adding ethylene glycol. Capto ™ MMC capacity was 260 U / ml resin. The capture step was performed using the following process.

Regenerate the column with 1-2 column volumes (CV) of 3 M NaCl, pH 10-12 at 300 cm / hr.
Equilibrate with 5 CV 50 mM sodium phosphate buffer (NaPi), 0.15 M NaCl pH 7.5 at 300 cm / hr.
Load a clarified undiluted sample (conductivity approx. 15 mS / cm) at 300 cm / hr.
• Wash 1: 4 CV equilibration buffer.
Wash 2: 0.9 C M 0.95 M NaAc, 5% (v: v) isopropanol, pH 4.9, 300 cm / hr.
Wash 3: 300 cm / hr of 4 CV equilibration buffer (up to a stable baseline, ie up to about 0.06 Au for a 5 mm flow cell).
Elute the product at a maximum of 120 cm / hr using 90 mM NaPi, pH 7.7 containing 6 CV 1.5 M NaCl, 40% ethylene glycol. Start recovery when absorbance increases (approximately 10 mAu from the new baseline). Collect about 4 CV.
• Regenerate the column as described above at 3 CV, downward flow direction, maximum 120 cm / hr.
・ Preferably in the upward flow direction, 3 CV H ₂ O, 3 CV 1 M NaOH (contact time of about 60 minutes), 2-3 CV phosphate buffer, pH about 7, 3 CV 20 mM phosphorus Perform in-place cleaning (CIP) and purification using sodium acid + 20% ethanol. Store in 20 mM sodium phosphate + 20% ethanol.

  Table 2 shows an example of a purification scheme. Table 3 outlines the process. FIG. 2 shows an example of a chromatogram in this step.

(Table 2) Capture step purification scheme: 13.5 x 2 ml (27 ml) XK 16 column packed with Capto ™ MMC (continued on next page)

Table 3 Summary of conditions in Capto ™ MMC capture stage

Example 3-Chromatography Intermediate Active Step Using Hydrophobic Interaction The product from the capture step comprising α-mannosidase is subjected to a hydrophobic interaction type resin such as a hydrophobic interaction type resin after the addition of sodium sulfate, e.g. It binds to Butyl Sepharose ™ 4 FF used in this example. The product elutes when the salt concentration is lowered. Its capacity was 195 U / ml resin. The following steps were used in the intermediate active stage.

Regenerate the column at 100 cm / hr using 1 CV of 20 mM sodium phosphate (NaPi) buffer, pH 7.5.
Equilibrate the column at 150 cm / hr using 5 CV 0.5 M Na ₂ SO ₄ , 20 mM NaPi, pH 7.5.
• Mix the pool of product from Step 1 with the same volume of 20 mM NaPi, 0.8 M Na ₂ SO ₄ , pH 7.5 and load it onto the column at 70 cm / hr. Mixing can be performed inline or up to 3 hours before the start of loading. A 1: 1 volume: volume (v: v) mixture corresponds to approximately 1.11: 1 weight: weight (w: w) (eluent: sodium sulfate buffer). If necessary, the conditioned load solution may be filtered through a 0.45 μm filter (hydrophilic PES or PVDF) prior to loading.
• Wash the column at 70 cm / hr with 3 CV equilibration buffer to remove ethylene glycol along with the host cell protein from the previous step.
Wash with 100 cm / hr using 3.5 CV of 20 mM NaPi, 1.2 M NaAc, pH 7.5.
• Wash with 150 cm / hr using 3.5 CV 0.6 M NaPi, pH 7.0.
Elute the product at 150 cm / hr using 4 CV 60 mM NaPi, pH 7.5. Collect approximately 2 CV peak from initial rise in absorbance to baseline.
Regenerate the column with 2 CV of 20 mM NaPi, pH 7.5, followed by 3 CV of H ₂ O at 150 cm / hr.
Cleaning and purification using 3 CV 1M NaOH (60 min contact time), 1 CV H ₂ O, 1-3 CV phosphate buffer, and 2 CV 20 mM sodium phosphate + 20% ethanol Process. Store in 20 mM sodium phosphate + 20% ethanol.

  Table 4 shows an example of a purification scheme. Table 5 outlines the process. FIG. 3 shows an example of a chromatogram in this step.

Table 4 Intermediate active purification scheme using Butyl Sepharose ™ 4FF packed in 13.5 cm x 2 cm ² (27 ml) XK 16 column (continued on next page)

(Table 5) Summary of conditions in Butyl Sepharose ™ 4FF process

Example 4-Chromatography Intermediate Passive Step Using Mixed Mode Ion Exchange Two eluates containing α-mannosidase from an intermediate active step are pooled and mixed 1: 1 with water (weight: weight) for conductivity. This was loaded onto a mixed mode ion exchange resin, such as ceramic hydroxyapatite I (CHT I) resin in this example. The product passes through unbound and the host cell protein binds to the column. The flow-through solution containing the product was collected. Its capacity was 550 U / ml resin. In the intermediate passive stage embodiment, the following steps were used.

Regenerate the column at 300 cm / hr using 2 CV 0.6 M NaPi, pH 7.0.
Equilibrate the column at 300 cm / hr with 5 CV 60 mM NaPi, pH 7.5.
-Load the adjusted eluate from step 2 at 300 cm / hr and collect the flow-through containing the product. The conductivity and pH of the load solution will be about 10 mS / cm and 7.3, respectively. Collect the flow-through containing approximately the product of the load volume and 2 CV wash from the OD rise to 20 mAu until the OD returns to 20 mAu. At the end of the process, the product pool is filtered through a 0.45 μm hydrophilic PES or PVDF filter.
• Wash the column at 300 cm / hr with 4 CV equilibration buffer.
Regenerate at 300 cm / hr using 3 CV 0.6 M NaPi, pH 7.0.
Clean and purify using 3 CV 1M NaOH (60 min contact time), 1 CV 60 mM NaPi, pH 7.5 and 2 CV 20 mM sodium phosphate + 20% ethanol. Store in 20 mM sodium phosphate + 20% ethanol.

  Table 6 shows an example of a purification scheme. Table 7 outlines the process. FIG. 4 shows an example of a chromatogram in this step.

Table 6 Intermediate Passive Step Purification Scheme Using CHT I Packed on a 10 cm x 2 cm2 (20 ml) XK 16 Column

(Table 7) Outline of conditions in CHT I process (continued on next page)

Example 5-Virus Inactivation Step Virus inactivation can be performed at different stages of the method. In this example, virus inactivation was performed after the intermediate passive step and before the refinement step. Virus inactivation of the intermediate passive pool containing α-mannosidase is carried out by incubation for 135 ± 15 minutes with 21% isopropanol (1: 1 mixture with 30% aqueous isopropanol) at 21 ± 5 ° C. The tank was cooled by a + 4 ° C. cooling jacket and the process was maintained at 21 ± 5 ° C. Isopropanol was removed using tangential flow filtration (TFF) using a 100 kDa polyethersulfone membrane Screen A (Millipore or Sartorius) and replaced with sodium phosphate buffer. In this example, the following steps were used for virus inactivation.

Mix the product from the intermediate passive process (flow-through solution) with 60 mM sodium phosphate containing 30% isopropanol in 1: 1 (v: v) equivalent to 1: 0: 94 (w / w) . For example, mixing is performed by supplying a recirculation pump. The protein concentration of the product will be about 0.3-1 mg / ml.
Incubate the solvent / product pool at room temperature for 135 ± 15 minutes.
Equilibrate TFF membrane with 60 mM sodium phosphate buffer.
• Transmembrane pressure (TMP) 1.1 bar, 21 ± 5 ° C., ultrafiltration with an inflow pressure of about 1.4 to 1.5 bar and an outflow pressure of about 0.7 to 0.8 bar. Concentrate to mg / ml).
• Replace approximately 6 volumes by diafiltration against 60 mM sodium phosphate buffer. Start at 1 bar TMP (1.4 bar inflow / 0.6 bar outflow). After one volume is exchanged, TMP can be raised to 1.1.
-Collect the non-permeate. Rinse the membrane with 2-3 system volumes of dilution buffer to remove loosely bound product. This rinse solution is collected together with the non-permeate. The final target protein concentration is 2 mg / ml (0.5-3 mg / ml).
Clean the membrane with H ₂ O followed by 0.5 M NaOH (60 minutes contact time). Store in 0.1 M NaOH.

  Table 10 shows the conditions in the virus inactivation / TFF step.

NaPi = 60 mMリン酸ナトリウム、pH 7.5 (Table 10) Summary of conditions for virus inactivation / TFF process

NaPi = 60 mM sodium phosphate, pH 7.5

Example 6-Chromatographic Refinement Process Using Anion Exchange Ionic Interactions with Anion Exchange Resin, eg Quaternary Ammonium High Performance Strong Anion Exchange Resin (Q Sepharose ™ HP Resin) in this Example The conductivity of the non-permeate containing α-mannosidase from the intermediate passive step is adjusted with the adjustment buffer (20 mM Tris-HCl, 10 mM NaCl, 75 mM mannitol, 0.005% tween ™ 80, pH 7.5). ). The non-permeate was diluted either directly or in-line before loading. The product was eluted in a container previously filled with 1 CV elution buffer by the addition of sodium chloride. Its capacity is 400 U / ml resin. The following steps were used in the refinement stage in this example.

Regenerate the column at 120 cm / hr using 1 CV 50 mM NaPi, 1 M sodium chloride, pH 7.5.
Equilibrate the column at 120 cm / hr with 5 CV 20 mM Tris-HCl, 10 mM sodium chloride, pH 7.5.
Load the diluted non-permeate from step 4 at 120 cm / hr.
• Wash the column at 120 cm / hr using 5 CV equilibration buffer and 1 CV 20 mM sodium phosphate, pH 7.5.
Elute the product at 120 cm / hr using 4 CV of 50 mM sodium phosphate, 0.2 M sodium chloride, pH 7.5 in a prefilled container (with 1 CV elution buffer). Collect the peak from 0.5% to 1.5 CV from the first increase in absorbance (starting recovery at 10-20 mAu) to 30-50 mAu (2 mm flow cell).
Regenerate the column at 120 cm / hr using 3 CV 50 mM NaPi, 1 M sodium chloride, pH 7.5.
• Clean and purify with 3 CV of 1 M NaOH (60 min contact time) and 3 CV of 10 mM NaOH. Store in 10 mM NaOH.

  Table 8 shows an example of a purification scheme. Table 9 outlines the process. FIG. 5 shows an example of a chromatogram.

Table 8: Example of refinement purification scheme using Q Sepharose ™ HP resin 19 cm x 2 cm ² (38 ml) (continued on next page)

(Table 9) Summary of conditions in Q Sepharose ™ HP process (continued on next page)

Example 7-Virus reduction process Virus reduction can be performed at different stages of the method. In this example, virus reduction was performed after the refinement process. The eluate from the refinement process is nanofiltered through a Planova ™ 15N filter after 0.1 μm prefiltration. The following steps were used.

• Prefilter the eluate from the refinement process through a 0.1 μm filter. The filter is rinsed with a small amount of 50 mM sodium phosphate, 0.2 M sodium chloride, pH 7.5 to remove the loosely bound product.
• Filter the eluate at 0.8 bar pressure and room temperature. This filtration is followed by a post-wash of approximately 3 volumes of the Planova 15 system using 50 mM sodium phosphate, 0.2 M sodium chloride, pH 7.5.

Example 8-Formulation and Storage
The buffer is exchanged for formulation buffer by tangential flow filtration (TFF) using a 100 kDa, Screen A, polyethersulfone membrane (Sartorius ™ or Millipore ™). By cooling the tank with a + 4 ° C cooling jacket, the process can be maintained at 21 ± 5 ° C. Approximate capacity is 100 l / m ^2. The following steps were used for formulation and storage.

Equilibrate membrane with 3.5 mM Na ₂ HPO ₄ , 0.17 mM NaHPO ₄ , 250 mM mannitol, 27 mM glycine, pH 7.7 (formulation buffer).
Dilute the purified product containing α-mannosidase to a target concentration of 2-3 mg / ml using approximately 1 volume of formulation buffer. If the protein concentration in the product is low, it is possible to reduce the volume before dilution with formulation buffer and concentrate to 4-6 mg / ml (but it is not essential).
Concentrate approximately 2 times to the target concentration of 6 mg / ml by ultrafiltration at TMP 0.8 and 21 ± 5 ° C.
• Replace 6 volumes by diafiltration against formulation buffer at TMP 0.8, 21 ± 5 ° C.
-Concentrate approximately 1.5 times by ultrafiltration and collect the non-permeate. Rinse the membrane with 1 system volume of formulation buffer to remove loosely bound product. This rinse solution is collected together with the non-permeate. An alternative is to measure the rinse at OD 280 and pool it only if it contains the product. The final target protein concentration is 7 ± 2 mg / ml.
Clean the membrane with H ₂ O followed by 0.5 M NaOH (60 min contact time). Store in 0.1 M NaOH.

(Table 11) Summary of conditions in prescription TFF process (continued on next page)

  The product was diluted to 5 mg / ml and sterile filtered. Fill bottle with filtered bulk and freeze.

Example 9-Semi-batch culture method for alpha mannosidase After thawing the cells, the cells were grown in shake flasks, 10 L seed bioreactors and 50 L seed bioreactors, after which they were produced in a production bioreactor (250 L). On the day of inoculation of the production bioreactor, the cell density in the 50 L seed bioreactor was between 2 and 2.5 MVC / mL. Cells were inoculated from the seed bioreactor into the production reactor at a cell density of 0.5 MVC / mL so that the cell suspension volume at the completion of inoculation was 100 L. The day of inoculation is called day 0, the next day is called day 1, and so on. From the first day to the last day of the experiment, feed medium was added daily during the boost at a predetermined rate (see below). From the first day to the last day of the experiment, glutamine and glucose were added daily during the boost at a predetermined rate and rule (see below). On the day when the viable cell density was ≧ 2.2 MVC / mL or the third day, whichever was earlier, the temperature was lowered to the production temperature.

（^*）第0日から第2日の間、pH制御のためにCO₂のみを自動添加した。アルカリは、pHが < 6.60になるまで添加しなかった。 (Table 12) Actual temperature and experimental conditions

( ^* ) From day 0 to day 2, only CO ₂ was automatically added for pH control. Alkali was not added until the pH was <6.60.

・ Base medium
ACF medium (ExCell302, SAFC) containing 11.1 mM (2 g / L) glucose, supplemented with 2 mM glutamine. The base medium without glutamine can be transferred to the production bioreactor and stored at 36.5 ° C. for up to 3 days, ie, during bioreactor sterility testing. Otherwise, store the medium at 4 ° C.

-Feed medium Feed medium E35 was a 35% CHO CD Efficient Feed B (InVitrogen catalog number SKU # A10240-01) feed concentrate diluted in ACF medium containing 11.1 mM glucose without glutamine. The feed medium was stored in the dark at 4 ° C. The feed medium can be maintained in the dark at room temperature for up to 96 hours, ie 4 days, during culture.

(Table 13) Volume of feed medium added per day

·Additive
a) Stock solution of 2500 mM glucose. b) 200 mM glutamine stock solution. c) Alkaline 0.5 M Na ₂ CO ₃ .

• Delivery of feed medium, glucose and glutamine During the boost, the feed medium was pumped daily. During the boost, glucose and glutamine were added daily in the following amounts of glucose and glutamine stock solutions to maintain their concentrations within the predetermined target range.

Rules for adding glutamine:
• Day 1 to Day 3: Add a volume of glutamine stock solution that results in a 2 mM glutamine concentration in the bioreactor.
-From day 4 onwards: Add a volume of glutamine stock solution that will result in a 1 mM glutamine concentration in the bioreactor.

Glucose addition rules:
• Day 0 to Day 8: Do not add glucose stock solution.
• After day 9: If the glucose concentration in the production bioreactor is ≦ 8 mM, add a volume of glucose stock solution that results in a glucose concentration in the bioreactor of 8 mM.

Overall picture of the method:
(Table 15) Daily overview of culture method

Example 10-Characterization of a purified product containing α-mannosidase Table 16 below shows that the purification scheme of the present invention has a higher proportion of 130 kDa glycoprotein species compared to degradation products of 75 and 55 kDa, respectively. It was shown that α-mannosidase was provided. The table also shows that the 250 L method using an improved washing step in both capture and intermediate active steps involving multimode ligands and a four-step purification method including Q sepharose HP refinement step yields yield, overall purity and 130 It shows that for all of the yields of kDa species, it gave better results than the 30 L method using the three-step method.

Table 16: Yields of α-mannosidase species in purified products

  The species distribution was confirmed in the HPLC diagram of FIG. 7 for the three methods, the table above representing the four-step method and the three-step method, respectively. The two-step method shown is one that does not use a multimodal ligand step. The first peak seen from the left is a 55 kDa species, followed by a 130 kDa species and a 75 kDa species, respectively.

References

Claims (10)

A method for purifying recombinant human lysosomal α-mannosidase from a cell culture, wherein a fraction of the cell culture containing recombinant human lysosomal α-mannosidase is subjected to chromatography with a resin containing a multimodal ligand, comprising: The multimodal ligand bound to the substance of formula (I), (II) or (III):

Where R of the substances of formula (II) and (III) is a functional group of formula (IV):

And a first eluate comprising recombinant human lysosomal α-mannosidase obtained from a resin comprising a multimodal ligand,
(i) applying a fraction containing recombinant human lysosomal α-mannosidase to a hydrophobic interaction chromatography resin to supply an eluate containing recombinant human lysosomal α-mannosidase;
(ii) passing a fraction containing recombinant human lysosomal α-mannosidase through a mixed-mode ion exchange resin to retain contaminants and supplying a flow-through solution containing recombinant human lysosomal α-mannosidase; and
(iii) A step of subjecting a fraction containing recombinant human lysosomal α-mannosidase to chromatography using an anion exchange resin to supply an eluate containing recombinant human lysosomal α-mannosidase
A method further comprising a method comprising:   2. The method of claim 1, wherein the fraction of the cell culture comprising recombinant human lysosomal α-mannosidase is a clarified undiluted harvest. The method according to claim 1 or 2 , wherein the fraction of the cell culture loaded on the resin containing the multimodal ligand is subjected to at least one washing step using a solution containing isopropanol. 4. The method of claim 3 , wherein the solution comprising isopropanol comprises at least 1% (V: V) isopropanol. The method according to any one of claims 1 to 4 , wherein the first eluate containing the recombinant human lysosomal α-mannosidase is eluted from the resin containing the multimodal ligand using an aqueous solution containing ethylene glycol or propylene glycol. . The method according to any one of claims 1 to 5 , wherein the recombinant human lysosomal α-mannosidase is selected from:
(A) a recombinant human lysosomal α-mannosidase having the sequence represented by SEQ ID NO 2;
(B) A recombinant human lysosomal α-mannosidase having a sequence having at least 90% sequence identity with SEQ ID NO 2 and having lysosomal α-mannosidase activity. a. On day 0, inoculating cells capable of producing recombinant human lysosomal α-mannosidase into a production reactor containing a base medium and supplying a cell culture;
b. adding a feed medium to the cell culture at least once from day 1;
c. adjusting the temperature of the cell culture to a maximum of 35 ° C. after day 3 or when the viable cell density exceeds 2.1 MVC / mL, whichever is first, and
d. A method for semi-batch or continuous production of recombinant human lysosomal α-mannosidase comprising the purification method according to any one of claims 1-6 . 8. The method of claim 7 , wherein the cell culture does not contain any auxiliary components derived from animals. 9. The method according to claim 8 , wherein the animal-derived auxiliary component is a cod liver oil auxiliary component. The method according to any one of claims 7 to 9 , wherein the recombinant human lysosomal α-mannosidase is selected from:
(A) a recombinant human lysosomal α-mannosidase having the sequence represented by SEQ ID NO 2;
(B) A recombinant human lysosomal α-mannosidase having a sequence having at least 90% sequence identity with SEQ ID NO 2 and having lysosomal α-mannosidase activity.

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